Calcitonin gene-related peptide and intermedin induce phosphorylation of p44/42 MAPK in primary human lymphatic endothelial cells in vitro

Background: Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide that belongs to the calcitonin family of peptides together with adrenomedullin (AM), adrenomedullin 2 (AM2), also known as intermedin (IMD), amylin (AMY) and calcitonin. Recent reports demonstrated the efficacy of drugs targeting the CGRP signalling axis for the treatment of migraine, but also revealed significant side effects such as inflammation and microvascular complications, including aberrant neovascularisation in the skin. These findings highlight the importance of studying cellular targets of CGRP in human tissues. Recent studies using animal models implicate the role of CGRP in lymphangiogenesis and lymphatic vessel function. However, whether CGRP has direct effects on lymphatic endothelial cells (LEC) remains unknown. In this study, we analysed phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) in proliferating primary human dermal LEC (HDLEC) stimulated with CGRP and compared with responses to AM2/IMD and AM. Materials and Methods: Primary breast HDLEC from two female donors (39 and 46 years old) were cultured in vitro. Cell viability and proliferation of sub-confluent HDLEC were confirmed by MTS assay and Ki67 immunostaining. Phosphorylation of p44/42 MAPK in HDLEC treated with CGRP, AM2/IMD or AM at different time points (1-30 min) and concentrations (10-12-10-6M) was assessed by immunoblotting. All experiments have been performed at least 4 times. Statistical analysis was done using Shapiro-Wilk and Kruskal Wallis tests followed by uncorrected Dunn's test, with p<0.05 interpreted as significant. Results: CGRP induced p44/42 MAPK phosphorylation in a time- and dose-dependent manner in proliferating primary HDLEC from both donors. p44/42 MAPK phosphorylation was induced by all three peptides (10-6M) at 5-15 min (p<0.05), but only CGRP-induced effect was sustained at 30 min. CGRP consistently induced p44/42 MAPK phosphorylation at 10-7-10-6M (p<0.05) in HDLEC from both donors, whilst responses to AM2/IMD and AM varied. Conclusions: Our study shows that CGRP and AM2/IMD have direct effects on proliferating primary HDLEC. These new findings reveal CGRP and AM2/IMD as novel regulators of lymphatic endothelial cell biology and warrant further investigation of their roles in pathologies associated with lymphatic function in the skin and other organs.